Effects of Glutathione S-Transferases (GSTM1, GSTT1 and GSTP1) gene variants in combination with smoking or drinking on cancers: A meta-analysis

Background: This meta-analysis aimed to systematically summarize the association between cancer risks and glutathione s-transferases (GSTs) among smokers and drinkers. Methods: Literature was searched through PubMed, Web of Science, CNKI, and WANFANG published from 2001 to 2022. Stata was used with fixed-effect model or random-effect model to calculate pooled odds ratios (ORs) and the 95% confidence interval (95% CI). Sensitivity and heterogeneity calculations were performed, and publication bias was analyzed by Begg and Egger’s test. Regression analysis was performed on the correlated variables about heterogeneity, and the false-positive report probabilities (FPRP) and the Bayesian False Discovery Probability (BFDP) were calculated to assess the confidence of a statistically significant association. Results: A total of 85 studies were eligible for GSTs and cancer with smoking status (19,604 cases and 23,710 controls), including 14 articles referring to drinking status (4409 cases and 5645 controls). GSTM1-null had significant associations with cancer risks (for smokers: OR = 1.347, 95% CI: 1.196–1.516, P < .001; for nonsmokers: OR = 1.423, 95% CI: 1.270–1.594, P < .001; for drinkers: OR = 1.748, 95% CI: 1.093–2.797, P = .02). GSTT1-null had significant associations with cancer risks (for smokers: OR = 1.356, 95% CI: 1.114–1.651, P = .002; for nonsmokers: OR = 1.103, 95% CI: 1.011–1.204, P = .028; for drinkers: OR = 1.423, 95% CI: 1.042–1.942, P = .026; for nondrinkers: OR = 1.458, 95% CI: 1.014–2.098, P = .042). Negative associations were found between GSTP1rs1695(AG + GG/AA) and cancer risks among nondrinkers (OR = 0.840, 95% CI: 0.711–0.985, P = .032). Conclusions: GSTM1-null and GSTT1-null might be related cancers in combination with smoking or drinking, and GSTP1rs1695 might be associated with cancers among drinkers.


Introduction
Cancer is one of the main reasons for the decline of life quality of people all over the world for its high mortality rate, and it brings great challenges to clinical therapy.A former study demonstrated that the number of patients diagnosed as cancer increased by 19.3 million and cases dying from cancer were up to 10 million worldwide in 2020. [1]Lifestyle factors such as smoking and drinking were considered as extremely influential stimulus of the occurrence of cancer.Polycyclic aromatic hydrocarbon, an emission of tobacco, has been regarded as the major organic pollutants affecting human health.A prospective study in 2018 suggested that smokers diagnosed with cancer might be associated with lower survival rate. [2][5] Acetaldehyde is the main toxic and harmful substance in the process of alcohol metabolism, which was classified as the group 1 of human carcinogens in the report of International Agency for Research on Cancer.Studies have shown that alcohol consumption increased cancer susceptibility via compromising human immune system and destroying immune mechanism, and this effect was more obvious in Asian populations. [6,7]lutathione S-transferases (GSTs) supergene family, mainly produced by liver, is one of the most important phase II enzymes in biotransformation in vivo.Each member of GSTs is located on different chromosomes and encoded by one or several highly polymorphic genes. [8]GSTs family plays an essential role in the defense mechanism that protecting against cytotoxic electrophilic chemicals, and GSTs can indirectly control some other metabolizing enzyme' activities. [3,9,10]Former studies have found that GSTs could reduce the cytotoxic effect by regulating chaperone proteins, ubiquitin-proteasome components, inflammation-related proteins, and apoptosis-related proteins. [11,12]In recent years, many studies have reported that lifestyle factors such as smoking and drinking might lead to changes in enzyme activity levels due to mutations of associated genes.Therefore, it is imperative to explore the mutual regulation and interaction between GSTs polymorphisms and cancers among smoking and drinking population.At present, GSTM1, GSTT1 and GSTP1 are the mainstream of research among the members of GSTs.GSTM1 is located on chromosome 1p13.3,encoding the u class of enzymes.GSTM1 can detoxifies cellular electrophilic substances by hormonally controlling under induction by phenobarbital and propylthiouracil. [13]GSTT1 is located on chromosome 22q11.2,encoding for θ class of enzymes.Similar to GSTM1, GSTT1 can be found in almost all eukaryotes and prokaryotes.The homozygous deletion mutations of GSTM1 and GSTT1 might lead to enzyme inactivation and alter the growth activity of certain tumor factors.Due to different coding sites of amino acids, GSTM1-null (GSTM1 -/-) and GSTT1-null (GSTT1 -/-) present the detoxification functional gene deficiency, thus altering susceptibility to some cancers aroused by environmental and lifestyle factors. [14]GSTP1rs1695(AA, AG, GG), located on chromosome 11q13, is the most studied gene encoding the π class of enzymes.GSTP1 polymorphisms were highly associated with alcohol consumption, drug-resistance and the development of cancer. [15]Some studies suggested that cancer risks differs significantly in patients with mutations of GSTs when smoking, drinking, ethnicity and source of controls were taken into account.For example, Katiyar et al, in 2020' study demonstrated GSTM1 mutations were associated with a higher incidence of cancer among smokers [16] ; however, ThekkePurakkal et al, in 2019, explained that there was no statistically significant difference in the effect of GSTM1 gene polymorphisms on cancer among smokers. [17]eta-analysis is a robust and scientific statistical analysis method based on huge data, which has incomparable advantages over other research methods, usually having a high credibility.In recent years, the relationship between various cancers and GSTs gene has been studied by scholars worldwide.Xavier et al, in 2017, found that Asian country people with GSTM1--null gene were more easily to develop gastric cancer than European and American. [18]Hernández et al [19] in 2017, demonstrated that GSTM1 and GSTT1 deletion could not be regarded as a separate factor influencing the survival of lung cancer.Lee et al [20] in 2020, showed that GSTP1rs1695 polymorphism was useful for the treatment of chronic myeloid leukemia patients.Hoxhaj et al in 2020, found that GSTM1, GSTT1 and GSTP1 polymorphisms might increase the risk of developing a second primary cancer among head and neck cancer survivors in different degrees. [21]However, because of the bias of language expression, regions, source and number of cases, there is still lack of a consistent conclusion.Therefore, a large scale of samples and suitable model designs are needed to further evaluate the relationship between GSTs gene and cancer development among smokers and drinkers.The aim of this study was to draw a latest conclusion on the relationship between GSTM1, GSTT1 and GSTP1 gene polymorphism and cancer risks among smokers and drinkers.Before us, no similar article has been found to systematically analyze the association between GSTs alone or in combination with smoking or drinking and all kinds of cancers, and we hope that these results will provide some insights into cancer prevention.

Literature search and selection criteria
Electronic literatures were searched by using the following databases: Web of Science, PubMed, WANFANG and CNKI.The keywords included (GSTM1 or GSTT1 or GSTP1 polymorphisms) and (smoking or cigarettes or tobacco) or (drinking or alcohol) and cancer by different combinations.The databases were searched in chronological order, from January, 2001 to the latest publication due date November, 2022.Only the casecontrols about the association between cancers related to GSTM1, GSTT1 and GSTP1 gene polymorphisms among smokers or drinkers that had been published in English or Chinese journals were kept.To reduce omissions as possible, we also searched and consulted references of relevant review articles and meta-analyses.To further investigate the relationship between the degree of smoking and cancer risks, smokers diagnosed with cancers were classified as light smokers (<20 packyear) and heavy smokers (>20 pack-year).
Articles that confirmed with the following criteria were included: Case-control study; Detailed data on the association of GSTM1, GSTT1 or GSTP1 polymorphisms with smoking and alcohol consumption were available for calculating the odds ratios (ORs) and estimating the 95% confidence interval (95% CI); The disease studied was clinically diagnosed cancer; and Full text available.
The reasons for exclusion were as follows: Review articles and meta-analyses as well as repeated articles in different databases; Minutes of meeting and clinical trials; No detailed data of case group and control group; and No original data on the association of GSTM1, GSTT1 or GSTP1 gene polymorphisms with smoking or drinking status.

Data extraction
After screening all articles according to the exclusion and inclusion criteria, we performed detailed data extraction for the articles including first author's last name, year of publication, ethnicity, country, source of control, cancer type, smoking or drinking status and genotype.For case-control studies on the same cancer published by the same author in different years, we kept the latest articles or the maximum sample size in principle.Two researchers used the same keywords to search articles independently.When an article contained unextractable data or some doubts, the 2 researchers would discuss together whether to keep this article.Subgroup analyses were conducted on the ethnicity (Caucasian, Asian, and mixed groups), the source of control group (hospital-based group and population-based group), and the types of cancer (lung cancer, liver cancer, bladder cancer, and so on) to calculate the differences in the prevalence of cancers, respectively.

Statistical analysis
The Stata software was applied to calculate the GSTM1-null/ presence, GSTT1 -null/presence, and GSTP1rs1695 GG + AG/ AA of the case group and control group among different smoking and drinking status.OR value and 95% CI were calculated to evaluate the association between GSTM1, GSTT1 and GSTP1 polymorphisms and cancer risks among smokers and drinkers.I-square and P value were used for the assessment of heterogeneity.If the I-square of the heterogeneity test was less than 50% (P > .05), it showed that the heterogeneity between studies was not statistically significant, and the fixed model should be used for calculation; otherwise, the random model would be applied.
In this study, year, ethnicity, and population origin were considered to be the sources that could influence heterogeneity, and the meta-regression analysis were used to find these variables.At the same time, combined with the use of sensitivity analysis, any study that had an impact on the overall results of the study could be identified.We also used the Begg and Egger's test to calculate possible bias between studies.All P values calculations were two-sided, and when P value < .05, it was considered to be statistically significant.
In order to improve the accuracy and credibility of the results of this experiment, we calculated the false positive reporting probability (FPRP) and the Bayesian false discovery probability (BFDP).As in previous studies, the threshold of FPRP was set to 0.2, and the prior probability was set to 0.25, 0.1, 0.01, 0.001 and 0.0001, to detect an OR of 1.5 associated with cancer risks in the study; and results with FPRP values less than 0.2 should be of concern. [22]Likewise, the BFDP results should be noted when the P value was less than 0.8. [23] Results

GSTT1 gene polymorphism studies among smokers and drinkers.
Of the 49 articles on smoking, 13 articles were for lung cancer, 6 articles were for bladder cancer, 5 articles were for gastric cancer, 5 articles were for cervical cancer, 4 articles were for liver cancer, 2 articles were for head and neck cancer, 2 articles were for breast cancer, 2 articles were for pancreatic cancer, 2 articles were for prostate cancer, as well as other 8 articles that were not performed subgroup analysis for the same reasons as above.Among subgroup of ethnicities, 30 studies were from Caucasian, 14 studies were from Asian, and 5 studies were mixed.
Of the 8 studies on drinking, 2 articles were for gastric cancer and 6 articles were for other cancers (breast cancer, thyroid cancer, esophageal cancer, colorectal cancer, lung cancer, and liver cancer).Among subgroup of ethnicities, 4 studies were from Caucasian, 2 studies were from Asian, and 2 studies were mixed (Table 5).

GSTP1 gene polymorphism studies among smokers and drinkers.
Of the 31 reports on smoking, 9 articles were for lung cancer, 4 articles were for bladder cancer, 4 articles were for gastric cancer, 3 articles were for head and neck cancer, 2 articles were for pancreatic cancer, and 9 articles without subgroup analysis for the same reasons as above.Among subgroup of ethnicities, 22 studies were from Caucasian, 6 studies were from Asian, and 3 studies were mixed.
Of the 5 studies on drinking, articles were for gastric cancer, head and neck cancer, esophageal cancer, and colorectal cancer; 4 studies were from Caucasian and 1 study was from Asian (Table 6).

Quantitative synthesis 3.2.1. The relationship between GSTM1 polymorphism and cancer risks alone and in combination with smoking
or drinking.GSTM1 (null/present) might rise the overall cancer risks in both smokers (I 2 = 68.20%,OR = 1.347, 95% CI: 1.196-1.516,P < .001)and nonsmokers (I 2 = 54.70%,OR = 1.423, 95% CI: 1.270-1.594,P < .001).And subgroup results demonstrated that GSTM1-null might increase cancer risks regardless of ethnicities (Caucasian and Asian) or whether smoking.Subgroup results also showed that high risk of lung and nasopharyngeal cancer could be found in both smokers and nonsmokers, but GSTM1-null could lead to increased cancer risks of the stomach, the cervical and the bladder even nonsmoking (Table 4).

The relationship between GSTT1 polymorphism and cancer risks alone and in combination with smoking or
drinking.Significant positive correlation was found between GSTT1 (null/present) and the overall cancer risks among smokers (I 2 = 79.80%,OR = 1.356, 95% CI: 1.114-1.651,P = .002)and nonsmokers (I 2 = 34.00%,OR = 1.103, 95% CI: 1.011-1.204,P = .028).Subgroup analysis found that smoking might increase the cancer risks in Caucasian smokers with a 1.318-fold and in Asian smokers with a 1.701-fold.The hospital-based source of control group was found with a 1.699-fold cancer risks among smokers, whereas, in contrast, the population-based nonsmokers were found with a 1.348-fold cancer risks.Subgroup results also showed that smoking was highly associated with cancer risks of the stomach, the pancreas and the prostate with a 1.944-fold, a 3.749-fold, and a 2.847fold, respectively, compared with nonsmokers (Table 5).
Significant association was also found between GSTT1 (null/present) and the overall cancer risks among drinkers (I 2 = 65.80%,OR = 1.423, 95% CI: 1.042-1.942,P = .026)and nondrinkers (I 2 = 373.30%,OR = 1.458, 95% CI: 1.014-2.098,P = .021).Subgroup analysis found that drinking might be more likely to increase the cancer susceptibility among Asian GSTT1-null carriers by 2.250 times compared with 1.543 times among Caucasians.GSTT1-null was associated with increased cancer risks among nonsmokers in both hospital-based and population-based control group.Subgroup results also showed that alcohol consumption might increase the cancer risks of the stomach to 1.877 times compared with nondrinkers (Table 5).

The relationship between GSTP1 polymorphism and cancer risks alone and in combination with smoking
or drinking.GSTP1 polymorphism (AG + GG/AA) was not statistically associated with the overall cancer risks in either smokers or nonsmokers.However, subgroup analysis indicated that GSTP1 (AG + GG) variations might reduce the risk of cancers among Caucasian smokers.And the negative correlation might also exit in the risk of head and neck cancer in combination with smoking (OR = .833,95% CI: 0.715-0.970,P = .019)(Table 6).

Publication bias
The results of publication bias for GSTM1, GSTT1, and GSTP1 gene polymorphisms and cancer risks were shown in Tables 4-6   respectively.Egger test for GSTT1 combined with smoking showed publication bias of 0.036 (Table 5).

Sensitivity analysis
No statistically significant variables were found in the sensitivity analysis of GSTM1, GSTT1 and GSTP1 gene polymorphisms.The meta-regression analysis found that the year, the ethnicity, and the source of control population were not associated with experimental heterogeneity.

FPRP and BFDP test
The FPRP and BFDP analysis values of GSTs gene polymorphisms and smoking or drinking were shown in Tables 4-6, respectively.According to the results of the FPRP analysis, a part of the results in GSTs polymorphisms models were noteworthy in the FPRP test at the OR of 1.5 with prior probabilities of 0.25 and 0.1, but very few results were noteworthy in the BFDP test at the OR of 1.5 with prior probabilities of 0.01, 0.001 and 0.00001, which suggested that the results of this study should be interpreted with caution and euphemism.

Discussion
Numerous studies have shown that smoking and drinking contributed to the development of cancers, but cancers did not occur in all smokers and drinkers.The occurrence of cancers had a certain probability which might closely relate to the genetic metabolism of genes.Smoking and drinking might have a synergistic effect with the biological metabolic enzymes regulated by GSTs genes.And it was shown in this study that GSTM1, GSTT1, and GSTP1 polymorphisms, alone or in combination with smoking or drinking, might affect the overall cancer risks differently, and the effects might be related to ethnicities.For GSTM1, plenty of meta-analyses have shown that GSTM1-null could increase cancer risks in some specific organs among smokers or drinkers [104][105][106][107][108] ; while some scholars suggested that this cancer susceptibility might not be augmented by smoking or drinking. [109,110]The outcome of this study indicated that GSTM1-null, both alone and in combination with smoking or drinking, was associated with the overall increased cancer risks.Moreover, subgroup analysis showed that cancer risks in GSTM1-null was organ-specific, especially for lung cancer and nasopharyngeal cancer among smokers, and liver cancer, head and neck cancer and stomach cancer among drinkers.In addition to smoking, the pathogenesis of lung cancer was affected by various factors such as living habits, environment, genetics, etc.The results reflected in this study showed that the risk of lung cancer was higher among nonsmokers (OR = 1.699,P < .001)than that among smokers (OR = 1.542,P < .001).Consistent with cumulative evidence, we also found that tobacco was not a contributing factor to stomach [52] ; cervical [46,111,112] ; and bladder [113] cancers.In studies of ethnic differences in GSTM1 gene polymorphisms, The evidence showed that the frequency of GSTM1-null in Asians was higher than that in Caucasians and the interaction of GSTM1-null and smoking increased the risk of oral cancer in Asians than Caucasians. [114,115]Our study showed that the overall cancer risks in GSTM1-null carriers were 1.515-fold among Asian and 1.312-fold among Caucasian smokers, respectively, suggesting that cancer risks related to GSTM1-null might be slightly higher in Asian smokers.But the interaction of GSTM1-null and drinking might increase cancer risks in Caucasian drinkers.In conclusion, GSTM1-null was associated with the overall increased cancer risks, especially in the lung, the bladder, the nasopharyngeal, the stomach and the cervical.And alcohol consumption might be a synergistic factor in promoting cancer risks of the liver, the head and neck and the stomach among GSTM1-null carriers.For GSTT1, one study in 2013 indicated that GSTT1 polymorphism might promote cancer development, especially in smokers. [104]But for cancer of different organs, many scholars put forward specific views.Du et al [106] found that smoking could rise the risk of esophageal cancer; Lao et al [107] found that drinking played a critical role in promoting the development of gastric cancer.The outcome in this study found that GSTT1-null combined with smoking or drinking could significantly increase the overall cancer risks (OR = 1.356 for smoking; OR = 1.423 for drinking).GSTT1-null combined with smoking might be obviously related to the pathogenesis of prostate cancer and pancreatic cancer for the risk being as high as 2.847 times and 3.749 times, respectively.Although Zeng et al [116] in 2016 found that alcohol consumption was not a co-factor of GSTT1-null in the development of gastric cancer, our findings suggested that an increased cancer risks might be associated with smoking (OR = 1.944,P = .045)and drinking (OR = 1.877,P = .021)among GSTT1 -null carriers.In addition, the risk of cancers increased by 1.318-fold and1.543-foldamong Caucasian smokers and drinkers, and by 1.701-fold and 2.250-fold among Asian smokers and drinkers, respectively.In conclusion, smoking and drinking were both synergistic factors in promoting the overall cancer risks among GSTT1 -null carriers.
[119] However, we noticed that Bao et al [120] stated that the interaction of GSTP1 gene mutation and smoking might reduce the risk of gastric cancer; and a huge review of Caucasian had estimated that smoking could modify lung cancer risks of GSTP1 gene mutation in Caucasians. [121]In this study, we observed that the GSTP1 (AG + GG) variations might act as a protective factor among nondrinkers (OR = .840,P = .032).In addition, the risk of head and neck cancer also decreased among smokers.Nevertheless, GSTP1rs1695 genetic variants might be negatively associated with cancer risks in Caucasian smokers or alcohol drinkers.However, due to the relatively small sample data, these conclusions should be treated with caution.
The limitations of this study were: The interactions between GSTs and other possible isozymes on cancer risks were not considered in this analysis.Cancers were caused by multiple factors; therefore, the synergistic or antagonistic effect of other factors should be considered as well as possible.Cancer risks might be more pronounced if the effects of genetic polymorphisms, alcohol and tobacco were combined together. [16,40]STs might have overlapping affinities, and that the combined effects of detoxification functions in GSTM1 and GSTT1 may have a greater impact on disease than the effect of independent one. [122]However, in the process of data extraction, some valuable articles could not be included in the analysis because of a lack of original data provided, resulting in the small sample size of data, especially for the analysis of the association between GSTs and cancers among drinking population.So, the sample data in this study was not available to further explore the relationship between the combined effects of gene-gene associations and smoking-drinking associations on cancers.Otherwise, the representativeness might be stronger to further explore the interaction between GSTM1, GSTT1 and GSTP1 polymorphisms and cancers among drinkers.
In conclusion, GSTM1-null might increase the risk of cancers, whether alone or in combination with smoking, but smoking and drinking might significantly contribute to increased cancer risks associated with GSTT1-null.No synergistic effect on the increased cancer risks was found between GSTP1rs1695 and smoking or drinking.And the role of GSTs polymorphisms in combination with smoking or drinking on cancers might be influenced by ethnicities.Broader sample data and appropriate experimental design were needed to further investigate the effects of GSTM1, GSTT1 and GSTP1 polymorphisms on cancer among smokers or drinkers.

Figure 1 .
Figure 1.The whole flow diagram of filtering the available articles in this study.

Table 1
Characteristics of the eligible studies for GSTM1 polymorphisms.
www.md-journal.comwere 24 articles for lung cancer, 8 articles for bladder cancer, 6 articles for liver cancer, 5 articles for gastric cancer, 5 articles for cervical cancer, 5 articles for head and neck cancer, 2 articles for esophageal cancer, 2 articles for nasopharyngeal cancer, 2 articles for breast cancer, and 2 articles for prostate cancer; the other types of cancer were not conducted subgroup analysis due to no more than 1 articles involved.Among subgroup of ethnicities, 34 studies were from Caucasian, 30 studies were from Asian, and 6 studies were mixed.

Table 2
Characteristics of the eligible studies for GSTT1 polymorphisms.
Study groups with more than one race or not explicitly mentioned.HB = hospital-based group, NA = not available, PB = population-based group.

Table 3
Study groups with more than one race or not explicitly mentioned.HB = hospital-based group, NA = not available, PB = population-based group.Characteristics of the eligible studies for GSTP1 polymorphisms.

Table 4
Integral analysis of the association between GSTM1 polymorphism and cancer risk among smoking and drinking populations. , Results shown in bold indicate the P value < .05, the statistically significant noteworthiness at 0.2 level by FPRP or 0.8 level by BFDP calculations.

Table 5
Integral analysis of the association between GSTT1 polymorphism and cancer risk among smoking ang drinking populations.

Table 6
Integral analysis of the association between GSTP1rs1695 polymorphism and cancer risk among smoking and drinking populations.Results shown in bold indicate the P value < .05, the statistically significant noteworthiness at 0.2 level by FPRP or 0.8 level by BFDP calculations.

Table 7
Comprehensive analysis of the association between GSTs and cancer risk.Results shown in bold indicate the P value < 0.05.